CHAPTER II
LITERATURE SURVEY
Chapter II Literature Survey
Nutrients play a vital role in the maintenance and functioning of an ecosystem.
Homeostasis of an ecosystem is maintained by recycling of organic matter within the
ecological niche brought about by the microorganisms present within the system.
Microorganisms, ubiquitous in nature, are the sole entities that bring about
biodegradation a process in which complex organic compounds are broken down to
simple utilizable compounds which ai e further recycled through biogeochemical
cycles (Pelczar et al., 1993).
2.1. SOURCES OF AROMATIC COMPOUNDS
2.1.1. Natural compounds
The major contribution to the input of the organic compounds is plant based,
which includes cellulose, hemicellulose and lignin which are glucosyl based except
for lignin which has an aromatic backbone structure. The benzene ring is the next
widely distributed structures in nature after the glucosyl residue (Diaz et al., 2001).
Other aromatic compounds present in nature include aromatic amino acids (tyrosine,
tryptophan, phenylalanine), alkaloids (nicotine, quinine, cocaine), hormones
(epinephrine, acetylcholine), vitamins (thiamine, biotin), steroids (Flavanoids,
quinones), pigments (chlorophyll), nucleic acids, etc. Commonly found aromatic
compounds at the plant roots include benzoate, phenols, 1-carvone, cymene, limolene
(Hegde and Fletcher, 1996; Gilbert and Crowley, 1997). All these compounds belong
to the aromatic homocyclic, heterocyclic and polycyclic ring structures. Lignin being
aromatic based polymer is the major contributor of aromatic compounds comprising
up to 25% of the land based biomass on earth (Diaz et al., 2001) and its recycling
along with other plant-derived aromatic compounds is vital for maintaining the
Earth's carbon cycle.
5
Chapter II Literature Survey
Some of the resistant aromatic compounds include tannins, a plant based
polyhydroxy aromatic compound. After lignin, these are the second most abundant
group of plant phenolics. The presence of a number of phenolic hydroxyl groups
enables them to form large complexes, mainly with proteins, and to a lesser extent
with other macromolecules like cellulose and pectin (Bhat et al., 1998). Other
naturally found aromatic compounds include crude oil which comprises of benzenes,
toluenes, ethylbenzenes, xylenes (BTEX), polyaromatic hydrocarbons and resins.
Though natural and being secluded, their introduction to the outer world has been due
to anthropogenic activities and since then has gained a lot of importance due to their
competitive involvement for degradation.
2.1.2. Anthropogenic compounds
Since industrialization, aromatic compounds are produced in large amounts
and are released into the environment by human activities. Various industries from
which the pollutants are released includes 1) chemical and pharmaceutical industries
that produce a variety of synthetic compounds and polymers, 2) paper and pulp
bleaching industries, 3) coal and petroleum industry 4) agricultural practices where
pesticides are extensively used (Diaz, 2004). In , 1988 the US Environmental
Protection Agency listed a number of chemicals as priority pollutants which included
pesticides, halogenated aliphatics, nitroaromatics, chloroaromatics, polychlorinated
biphenyls, phthalate esters, polycyclic aromatic hydrocarbons and nitrosamines
(Fewson, 1988). Certain structural elements such as halo or nitro -substituents are rare
in naturally occurring compounds. Fluoro- compounds particularly perfluoroalkyl,
sulfo and azo groups, which are structural features of technically relevant commodity
Chapter 11 Literature Survey
chemicals, are practically unknown amongst natural products and can be considered
as real xenophors (Reiger, et al., 2002). Gribble, (2003), has reviewed the presence of
various organohalogenic compounds in nature produced by diverse species such as
marine plants, marine sponges, bacteria and fungi, plants, algae, lichens, terrestrial
plants, animals and humans. Other abiogenic methods of introduction of these
compounds include biomass fires, volcanoes, and other geothermal processes.
Nitroaromatic compounds are used worldwide as explosives, pesticides, and as
precursors for the manufacture of many products, including dyes, pharmaceuticals,
and plastics. These compounds do not only come from man-made sources but are also
formed by some natural processes, such as photochemical reactions in the
atmosphere. Nitroaromatic compounds are well known as toxins; some are mutagenic
and/or carcinogenic and others are uncouplers of cellular phosphorylation reactions
(Crawford, 1995).
Xenobiotic compounds (organohalogens such as polychlorobiphenyls (PCB)
Dichloro-Diphenyl Trichloroethane (DDT), are recalcitrants and their lipophilic
property enhances their bioaccumulation and biomagnification (Vettery, 2002; Gray,
2002; Goerke et al., 2004; Richter and Nagel 2006; Nfon and Cousins, 2006). Though
the concentration of xenobiotics is present in sub-lethal levels, their long-term
exposure causes significant damage to marine populations and may be carcinogenic,
mutagenic or teratogenic. Animals such as seals, bald eagle, and seabirds showed
disrupted hormonal cycle, leading to reproductive dysfunction such as reduction in
fertility, hatch rate, alternation of sex behavior and viability of offspring (Crews et al.,
1995). Due to long environmental and biological half lives, recovery from the effects
of many xenobiotic compounds is expected to be slow. Indeed, it has been shown that
7
Chapter II Literature Survey
more than 15 yrs are required to remove the negative effects of DDT on reproduction
of white tail eagles (Haliaeetus albicilla) in the Baltics, and another 10 years for the
population to recover. Likewise, long recovery times have been reported for harbour,
grey and ringed seals in the Baltic. The grey seal (Halichoerus grypus) population in
the northern Baltic has shown marked increase since the ban of DDT and PCB in the
Baltic region (Wu, 1999).
Degradations of such compounds are brought by microorganisms occur either
through metabolic or cometabolic processes.
2.2. METABOLISM / COMET4BOLISM (Grady, 1985)
Microorganisms can use aromatic compounds as a source of carbon and
energy or they may be biotransformed with reduction of toxicity or to an inactive
form. Such metabolism normally occurs in the presence of additional carbon sources,
which supports the growth of the organism and simultaneously degrade them. Thus is
the biotransformation of the additional aromatic compounds present in the medium.
In some metabolic cases the organic compound is similar to a substrate and therefore
gets metabolized by a mechanism called gratuitous.
With various pathways present in nature to bring about degradation of
aromatic compounds, many microorganisms utilize these compounds as sole source of
carbon based on the activity of enzymes. Xenobiotic compounds, due to their
complexity and uniqueness, tend to remain in the environment for long periods of
time. These compounds are called persistent if they are biologically degraded at a
8
Chapter II Literature Survey
2004), is widely distributed in nature is not utilized under anaerobic condition in the
absence of nitrates. Kesseru et aL, (2005), has shown the dependence of the anaerobic
bacteria Pseudomonas butanovorans to use salicyclate as electron donor for nitrate
reduction. This was not achieved in the presence of sulfates or phosphates even at
high concentrations. Glucose abundantly present in nature also proves to play an
important role in the degradation of certain xenobiotic compounds. Tharakan and
Gordon, (1999), showed that Trinitrotoluene (TNT) a chemiCal used in explosives has
been placed as priority chemical list by the US government, can be significantly
removed in the presence of glucose by bacteria. Bacteria isolated from the TNT
contaminated soil could bring about 100% transformation, which was only partially
removed in the absence of glucose to about 38% of the initial concentration. Glucose
also facilitated a Pseudornonas sp. (Ziagova and Liakopolou, 2007), in the
degradation of 1,2-Dichlorobenzene a known xenobiotic without which it hardly
grew. Raymond and Alexander, (1971), has shown that m-nitrophenol resistant
bacteria utilized it only in the presence of p-nirophenol, which was used as a source of
carbon and energy. Benzopyrene, one of the polyaromatic hydrocarbons (PAH) was
seen to be removed when Sphingomonas JAR02 was incubated with benzopyrene in
the presence of root products. The cometabolism of the benzopyrene was facilitated
during the utilization of other aromatic compounds present (Rentz et al., 2005). Van
Herwijnen et al. (2003), indicated that the isolate Sphingomonas LB216 could
cometabolise various PAHs such as phenanthrene, fluoranthene, anthracene,
dibenzothiophene only when initially grown in the presence of fluorene.
9
Chapter II Literature Survey
2004), is widely distributed in nature is not utilized under anaerobic condition in the
absence of nitrates. Kesseru et al., (2005), has shown the dependence of the anaerobic
bacteria Pseudomonas butanovorctris to use salicyclate as electron donor for nitrate
reduction. This was not achieved in the presence of sulfates or phosphates even at
high concentrations. Glucose abundantly present in nature also proves to play an
important role in the degradation of certain xenobiotic compounds. Tharakan and
Gordon, (1999), showed that Trinitrotoluene (TNT) a chemical used in explosives has
been placed as priority chemical list by the US government, can be significantly
removed in the presence. of glucose by bacteria. Bacteria isolated from the TNT
contaminated soil could bring about 100% transformation, which was only partially
removed in the absence of glucose to about 38% of the initial concentration. Glucose
also facilitated a Psendortionas sp. (Ziagova and Liakopolou, 2007), in the
degradation of 1,2-Dichlorobenzene a known xenobiotic without which it hardly
grew. Raymond and Alexander, (1971), have shown that m-nitrophenol resistant
bacteria utilized it only in the presence of p-nirophenol, which was used as a source of
carbon and energy. Benzopyrene, one of the polyaromatic hydrocarbons (PAH) was
seen to be removed when Sphingornottas JARO2 was incubated with benzopyrene in
the presence of root products. The cometabolism of the benzopyrene was facilitated
during the utilization of other aromatic compounds present (Rentz et al., 2005). Van
Herwijnen et al. (2003), indicated that the isolate Sphingomonas LB216 could
cometabolise various PAI-Is such as phenanthrene, fluoranthene, anthracene,
dibenzothiophene only when initially grown in the presence of fluorene.
1 0
Chapter II Literature Survey
Cometabolism and gratuitous metabolism thus play a very important role to
bring about biotransformation of various xenobiotic compounds which otherwise
would only be persistent or recalcitrant.
In nature, microbial biotransformation and metabolism of aromatic rings is
found to occur by cleavage of the aromatic nucleus via catechol by different pathways
as elucidated.
2.3. AROMATIC RING CLEAVAGE PATHWAYS
The most abundant aromatic nucleus encountered in the environment is that of
benzene, most stable but enzyme labile. For an enzyme to cleave the benzene ring, it
is a prerequisite to add two molecules of oxygen in to the ring to convert it to a
dihydrodiol product. Aromatic acids such as benzoates, biphenyls, etc. required the
addition of two oxygen atoms in. the ring, while, monohydroxylated aromatic
compounds such as phenols, hydroxybenzoates required only one oxygen atom to be
added in the ring.
The fission of the aromatic ring takes place by two major mechanisms. An
intradiol fission wherein the bond between the two vicinal hydroxyl groups is broken
known as the ortho -cleavage pathway or by extradiol fission where the cleavage takes
place adjacent to either of the hydroxyl groups called meta -cleavage pathway. A third
kind of ring cleavage is seen where the p-hydroxydiol compounds are broken is called
the gentisate pathway. The breakdown is not ortho due to para- positioned hydroxyl
group, but is similar to meta-cleavage.
Chapter II Literature Survey
Most of the aromatic compounds that undergo biodegradation converge to
catechol (Cerdan et al., 1994; Ngai et al., 1990) (fig. 2.1), protocatechuate (Noda et
al., 1990) (Fig. 2.2), gentisate (Tomasek and Crawford, 1986; Stolz et al., 1992) or
homogentisate (Hagedorn and Chapman, 1985; Fernandez-Canon and Penalva, 1998)
(Fig. 2.3) or their derivative s .
2.4. CLEAVAGE PATHWAYS OF SUBSTITUTED AROMATIC COMPOUNDS
In the environment large number of compounds are seen with different
molecular structures, some of these compounds have a basic aromatic ring with
substitutions which are hydrogenated, nitrated, chlorinated or have heterocyclic ring
with the incorporation of either oxygen, nitrogen or sulphur in the aromatic ring.
Many of such compounds are known to be biotransformed by a wide variety of
microorganisms. However, degradation of nitrogen containing monocyclic aromatic
compounds are explained herewith.
2.4.1. Nitroaromatic compounds
Nitroaromatic compounds constitute a major class of widely distributed
environmental contaminants. Compounds like nitrobenzene, nitrotoluenes,
nitrophenols, nitrobenzoates and nitrate esters are of considerable industrial
importance. They are frequently used as pesticides, explosives, dyes, and in the
manufacture of polymers and pharmaceuticals. Many nitroaromatic compounds and
their conversion products have been shown to have toxic or mutagenic properties.
Most of them are biodegradable in nature by various microorganisms. However, most
12
Meta- cleavage catechol 2,3-dioxygenase ..0„...",....,,,e
Ortho- cleavage
catechol 1 ,2-dioxygenase %,....N.,.....,...4.,
COOH ••■• COOH
OH
Fig. 2.1: Ortho- and meta- cleavage of Catechol
0 2-Hydroxy-cis,cis-
muconate semialdehyde
2-hydroxy-muconate formate semialdehydehydrolase
CH2 COOH
cis-2-Hydroxypenta -2,4-dienoate
cis,cis-Mucon ate
itarticonate cyclo-isomerase
COOH
Y ° Muconolactone
1 dienelactone hydrolase
If2-oxopent-4-enoate hydratase ...., COOH
Maleylacetate
COOH
CH3 COON
4-Hydroxy -2-oxovalerate
1, 4-hydroxy- 2-oxo-valerate aldolase
Pyruvate + Acetaldehyde
Ifmaleyl-acetate reductase
000011
COOH
13-ketoadipate
3-oxoadipate CoA-transferase
O CSC0A COOH
acetyl-CoA C-acyltransferase
Succinyl CoA + Acetyl CoA
Ortho- cleavage
protocatachuate 3,4-dioxygenase OH
• protocatechuate / 3,4 -dihydroxy benzoate
Meta- cleavage
proto-catechuate 4,5-di- oxygenase
OH
Fig. 2.2: Ortho- and meta- cleavage of protocatachuate
3 -Carboxy -cis,cis-muconate 4-Carboxy-2-hydroxy muconate semialdehyde
HO COOH c-:0
COOH
Gentisate 1,2- dioxygenase
Maleylpyruvic acid
hornogentisate 1,2-dioxygenase
Fig. 2.3: Gentisate/homogentisate pathway
Gentisic acid
Homogentisate pathway
Homogentisate
OH
Maleylpyruvate isomerase
COOH , c -z.0
Fumatylpyruvic acid
COOH
0 H
H
cooil 0
MalcOacetoacetic acid
COOH
Acylpyruvate aldolase
Pyruvate fumerate
maleyl-aceto-acetate isomerase
COON
HOOC
H 0
4-Fumarylacetoaeetate
fumaryl-aceto -acetase
fumerate ticetotteetate
Chapter 11 Literature Survey
contaminated environments have combinations of nitroaromatic compounds present,
which complicates the bioremediation efforts (Ye et al., 2004).
Bacteria appears to have evolved four main strategies to address the nitro-
group under aerobic conditions (Nishino et at, 2000): (a) dioxygenation of the
nitroaromatic ring, with release of the nitro-group as nitrite and production of
dihydroxy intermediates, (b) monoxygenation to epoxides, (c) formation of a
Hydride-Meisenheimer 'complex and (d) partial reduction of the nitro -group,
formation of hydroxylaminobenzene derivatives and ammonia release, followed by
rearrangement of the hydroxylaminobenzene to the corresponding catechol and
elimination of another ammonia molecule.
The aromatic 7 electron nucleophilic mechanism with the additional nitro (-
NO2) electron withdrawing property protects nitroaromatics tlrom initial attack by
oxygenases but is favourable for reductive attack (Rieger & Knackmuss, 1995). On
the other hand, anaerobic reductive attack produces the aromatic amine ( —NH2), an
electron-donating group which represents a barrier to further attack by anaerobes
(McCormick et al., 1976). Thus, nitroaromatics often either persist or become amino
end products in the environment.
2.4.1.1. Nitrobenzene
Nitrobenzene is the simplest of all aromatic nitrates used in the manufacture of
rubber, drugs, dyes, pesticides, lubricating oils, etc.
The most common widespread method of degradation of these compounds is
either by partial reduction of the nitro group or by dioxygenase pathway (Ye, 2004).
13
Chapter II Li terature Survey
Nishino et al., (2000), reported the pathway followed by Pseudomonas
pseudoalcaligenes (Fig.2.4) through partial reduction of nitrobenzene by enzyme
nitrobenzene reductase to give nitrosobenzene which is further reduced by reductase
to give hydroxylaminobenzene. Enzyme mutase rearranges the hydroxylatnino group
to amine and hydroxyl at subsequent positions on the ring. Somerville (1995), has
reported that Pseudomonas putida carried out these steps followed by ring cleavage
brought about by aminophenol dioxygenase. Intermediates 2-amino muconate and 2-
amino-2,4-penteneoate was found to undergo deaminase reaction to form 2-oxo-3-
hexene -1,6-dioate and 2-oxo-4-penteneoate (He et al., 1997), which are
intermediates of the m-cleavage of catechol pathway. The nitrobenzene dioxygenase
enzyme produced by Cornamonas sp. yielded catechol by the loss of nitro group
(Nishino et al., 1995). Catechol breakdown leads to form metabolites which are
utilized in the TCA.
• Nitrobenzene can be converted to aniline under anaerobic condition or can be
reduced to aniline under aerobic condition as shown by the reaction steps via
hydroxylaminobenzene.
2.4.1.2. Nitrophenol
Degradation o, m & p, forms of nitrophenols are discussed here. 2-nitrophenol
has shown to have the simplest degradation pathway in which the nitro group is
removed by the action of 2 -nitrdphenol 1,2 -dioxygenase giving catechol. Catechol
proceeds towards the ring fission and intermediates utilized in TCA (follow
downstream pathway from catechol in fig.2.5 - degradation of nitrobenzene) and the
14
anaerobic
catechol
OH
hydroxylamino benzene
Ilt mutase
catechol-2,3-dioxygenase
2-hydroxymuconie isomerase
2-hydrox-ymueonie
0
2 -oxo-3-hexene -I,6-dioate
COOH
COOH
COOH
••-■, COOH
deearboxylase N H2
COOH 2-aminomuconic COOH
1 decarboxylase
2-oxo-4- COOH penteneoate
CH2
NH2
COOH
CH2
2-amino-2,4- penteneoate
hydroxylase
4-hydroxy-2 COOH -oxovalerate
HO CH3
aldolase
Acetaldehyde + acetic acid
Fig. 2.4: Degradation pathway nitrobenzene
NBdio),:ygenase/
OH
OH
nitrobenzene NB reductase
NHOH
Aniline
2-hydroxymuconic semialdehyde •••.,
COOH
CHO
dehydrogenase
OH Aminophenol- 2,3 -dioxygenase
NH2
' L-''' COOH 2-aminomuconic
..`kz.., „CHO semialdehyde
dehydrogenase
2-amino phenol
NO2 NO2
monooxygenases
HO 3-ni trophenol
NHOH
HO NO2 HO 3-hydroxyaminophenol
4-nitroresoOrcmH ol
OH catechol
1 reductase
4-hydroquinone
HO NO2
HO 4-nitrocatechol
OH OH HO
dioxygenase
COOH
COOH
maleylacetate
Fig. 2.5: Degradation pathway of nitrophenols
mutase dioxygenase
monooxygenases hydroxyl amino lyase
OH Alt
HO COOH CHO
fl-hydroxymueonie semialdehyde
HO
HO 1 ,2,4-benznetriol
HO aminohydroquinone
Ring fission
Chapter II Literature Survey
organism that followed such a pathway reported by Zeyer el aL (1985), was
Pseudomonas putida.
3 -nitrophenol was seen to have broken down by two other pathways by
different organisms (studies carried out by different authors). P. putida B2 partially
reduced 3-nitrophenol to 3-hydroxyamino phenol followed by an addition of two
hydroxyl groups, a reaction catalysed by 3 -hydroxyamino phenol -3,4 -dioxygenase to
give 1,2,4 -benzenetriol (Meulenberg, 1996). Organism Ralstonia eutropha
transformed 3 -hydroxyamino phenol to amino hydroquinone by the enzyme 3 -
hydroxyamino phenol mutase (Schenzle, 1997). Further reactions cleave amino
hydroquinone which is then utilized for cellular purposes.
Three different pathways were found to follow during the breakdown of 4-
nitrophenol. Bacterial species such as Pseudomonas (Chauhan et al.,. 2000),
Moraxella (Spain et al., 1991), Bacillus (Kadiyala, 1998), Arthrobacter (Hanne et al.,
1993) were found to follow different pathways to transform 4 -nitrophenol. The major
route being their conversion to 4 -hydroquinone followed by ring cleavage to give 13 -
hydroxymuconic semialdehyde. Enzyme monoxygenase catalysed the reaction of
hydroxylating at 2- and 3- positions of 4-nitrophenol to give catechol and resorcinol
respectively in species Bacillus and Arthrobacter. Further hydroxylation and removal
of nitro group gave 1,2,4 -benznetriol which is ultimately cleaved by.dioxygenase to
give linear compounds easily utilizable by bacteria.
2.4.1.3. Nitrotoluene degradation
Mono and di-Nitrotoluenes contain single or two nitro groups in benzene ring
with methyl group as a parent compound. Degradation of the mono-nitrotoluenes has
15
Chapter II Literature Survey
been shown to follow three pathways (Fig.2.6). Parales, et al. (1998), proposed that
Pseudomonas species converted 2-nitrotoluene to 2-methyl catechol which proceeds
with ring cleavage. In case of 4-nitrocatechol, two pathways were seen to have
followed; Pseudomonas strain TW3 and 4NT (James et al., 1998) followed an initial
hydroxylation of the methyl group by hydroxylases then further oxidizing until 4-
nitrobenzoate was formed. Further, partial reduction of the nitro group lead to
conversion of 4-hydroxyaminobenzoate to 3,4-dihydroxycatechol by dioxygenases,
which was further cleaved by enzyme dioxygenases. The other pathway shown by
Mycobacterium HL4NT-1, includes partial reduction of nitro group not altering the
methyl group. Enzyme reductase catalyzed the conversion of nitrotoluene to
hydroxylaminotoluene to aminocresol by mutase followed by aromatic ring cleavage
by dioxygenases.
Dinitrotoluene (2,4-Dinitrotoluene and 2,6-Dinitrotoluene) are found to follow
different modes of breakdown (Fig. 2.7). Breakdown of 2,4-Dinitrotoluene by
Pseudomonas was studied by Suen et al. (1993). Reaction flows from denitrification
by dioxygenases to form 4-methyl-5-nitro-benzene-1,2-diol, followed by
monooxygenases converting this compound to a quinone by replacing the remaining
nitro group with oxygen, which gives methyl-benzenetriol. The benzenetriol becomes
available for ring cleavage by dioxygenases. 2,6-Dinitrotoluene is also initially
attacked by dioxygenases replacing a nitro group with two vicinal hydroxyl groups to
give 3-methyl-4nitrocatechol. The dihydroxy compound becomes vulnerable to the
dioxygenase enzyme attack. This pathway was proposed by Nishino et al., (2000) in
organisms Burkholderia cepacia and Hydrogenophage paleronii.
16
02N
p-Nitrotoluene
COOH 02N
ON
HOHN COOH 4-hydroxylamino benzoic acid
Dioxygenase
HO COOH
3,4-dihydroxy HO benzoate
HOHN CH3
p-Hydroxylamino toluene
02N CHO
4-nitrobenzaidehyde
hydroxylase
mutase
CH3
H2N
2-Amino-5-methyl -6-0xo-hexa-2,4-
dienoic acid
H2N 2-Amino-5-
methyl-hexa- 2,4-dienedioic acid
CH3
COCH3
COOH
HO 2-hydroxy-6- oxohept a-2,4- dienoate
Fig. 2.6: Degradation pathway of Nitrotoluenes
hydroxylase 02N
4-nitrobenzylalcohol
hydroxylase
reductase
HO OH 2-methylcatechol
CM2 2-hydroxy penta-2,4-
COOH dienoate
HO
CH2OH
OHC
HOOC COOH
ct-hydroxy-y-
HO carboxymuconic semialdehyde
TCA
02N
NO2
dioxygenase
CH3
NO2
2,6-dinitrotol tame
dioxygenase
2,4-dinitrotoluene
NO2 4-Methyl-5
-nitro-benzene- 1 ,2-diol HO
CH3
NO2 3-methyl-4-nitrocatechol
HO
HO
monooxygenasc
OH 5-Methyl-benzene-
1 2,4- tri HO
HO
Fig. 2.7: Degradation pathway of Din itrotoluenes
OH dioxygenase
dioxygenase
OH
dioxygenase
CH3
NO2
2-hydroxy-5-nitro-6- oxohepta-2 ,6-
di enoic acid
COOH CHNO2 2-1 lydroxy-5-
H 0 nitro-penta-2,4- dienoic acid
OHC'
HOOC
OH
H 2,4-Dihydroxy-5-methy1-6-oxo-
hexa-2,4-dienoic acid
TCA .
Chapter II Literature Survey
2.4.1.4. Nitrobenzoate degradation
Three different nitrobenzoates are found based on the placement of the nitro
groups on the parent benzoate (Fig.2.8). 2 -nitrobenzene is converted to 2-
hydroxylaminobenzoate by undergoing a reductive degradation in aerobic condition
(Durham, 1958; Chauhan et al., 2000; Hasegawa et al., 2000). Pseudomonas
fluorescens further carried out transformation to produce 3-hydroxy-2-anthrani late
catalyzed by enzyme mutase, which followed a breakdown reaction by dioxygenases.
In Athrobacter protophormiae, further reductive reaction was carried by converting
hydroxylamino group to form 2-anthranilic acid. Anthranilate dioxygenase released
the amino group from the ring which made the compound susceptible to ring
cleavage.
Formation of protocatechuate from 3-nitrobenzene was catalyzed by
dioxygenase in Pseudomonas or it may be sequentially hydroxylated as observed in
Nocardia species (Cartwright et al, 1958) to give 3-hydroxybenzoate before the
formation of protocatechuate. Protocatechuate was then available for ring cleavage.
4-nitrobenzene was partially reduced to nitrosobenzene before further
transformation could be carried out; Various pathways were observed to have
followed from this compound. Comamonas acidovorans, P. picketii and P. putida
were seen to partially reduce nitrosobenzene to give hydroxylaminobenzoate which
was then converted to protocatechuate by releasing amino group by the enzyme
hydroxylaminolyase. Burkholderia and Ralstonia partially converted
hydroxylaminobenzoate to protocatechuate but the major conversion was to 2 -
hydroxy-p-aminobenzoate and 3-hydroxy-4-acetoamidobenzene formed the action of
mutase on 2-hydroxy-p-aminobenzoate.
17
COOH
NO2
2-nitro benzoate
COOH
NO
2-nitroso benzoate
OH COOH
OH
5
as
CD
NHOH
COOH
02N 3-nitro benzoate
COOH 02N
Crc Cr0
COON
HO
/4"71 COOH
NHOH p-hydroxylamino benzoate
NH2 13-ketoadipate
0
F,;
COOH
OH 3-hydroxy-4- 0,„ NH aeetamidobenzoate
Fig. 2.8: Degradation pathway of nitrobenzoates
4-nitro benzoate
ic I.
CD
NO
a CD
COOH
COOH p-hydroxy benzoate
COOH
HO prptoeateehuate
/ OHC HOOC
HO
COOH
OHC / COOH HOOC
COOH
Chapter II Literature Survey
Complete reduction of the nitroso group of the 4-nitrosobenzoate gave p-
anthranilic acid which was converted by enzyme p -anthranillic aminolyase to give
protocatechuate. Protocatechuate is then normally broken down by various organisms
for their source of carbon and energy.
2.4.2. Degradation of anilines
Anilines are usually easily metabolisable and are utilized by various
soil borne micro-organisms. But it has been revealed that with addition of a
substituent group to the aromatic ring, its susceptibility to bacterial degradation
reduces. Paris et al. (1987), has shown that aniline is the most easily metabolisable
compound but insertion of methyl, chloride, bromo, methoxy, nitro or cyano groups in
the aromatic ring increases its resistance to degradation by micro -organisms. The rate
of transformation of these compounds decreased in the order. aniline > 3 -bromoaniline
> 3 -chloroaniline > 3 -methylaniline > 3 -methoxyaniline > 3 -nitroaniline > ' 3 -
cyanoaniline. Other simple forms of anilines include aminophenols,
chloroaminophenols, aminobenzoates and chloroanilines.
Besides their biodegradability, aromatic amines are also gaining importance
for their carcinogenic properties. 80 different aromatic amines were tested for their
mutagenecity by carrying out AMES test involving various strains of Salmonella
typhimurium (Chung et al., 1997). A transformed product of an azo dye, an aromatic
amine p-phenylenediamine, which is extensively used in hair dyes, was found to be
the most potent carcinogen. According to Chung, Crebelli et al. (1989), has reported
that p -phenylenediamine is not a direct or weak carcinogen but gets transformed to a
potent carcinogen when activated by the action of enzymes within a biological entity.
18
Chapter II Literature Survey
Many aromatic amines have been found to be candidates of potent mutagens
on enzymatic induction and the main criterion for the aromatic amines to be
carcinogenic is based on their ability to form nitrenium ion (Wild, 1990). Chung
concluded from his study that diamino aromatic compounds with distal placement of
amino groups showed highest mutagenicity. Such compounds with other groups such
as nitro or large alkyl groups placed at its vicinity were found to be less mutagenic.
Compounds such as aniline, m and p-aminophenol, were found to be non toxic but in
late 19th century, carcinogenicity in workers (urinary bladder cancer) at a dyestuff
industry was related to aromatic amine toxicity. Further, Weisburger (2002), reported
that 2-aminofluorene was tested to show a positive reaction towards carcinogenicity
in mice after activation by cytochrome P450 present in its liver. Aromatic amines
therefore have been gaining importance as the intermediates formed during the
transformation of complex aromatic amino compounds could be a potent carcinogen.
Several workers have reported the degradation of aniline by various organisms
(Anson et al., 1984; Peres et al., 1998; Vijay Shanker et al., 2006; Liu et al., 2002).
Catechol has been reported as most common intermediate during the degradation of
aniline, further breakdown of catechol could proceed the catechol-1,2 or catechol,2-3
diox-ygenase pathway for complete mineralization. Lyons et al., (1984) proposed
various interactions of aniline in the environment, which explained its degradation
and polymerization (Fig. 2.9) and has reported the aniline transformation via catechol,
1,2-dioxygenase.
Degradation of 2-aminophenol has been reported in Pseudomonas putida
HS12 by Park et al., (2001); and in Pseudomonas sp. AP-3 by Takenaka et al.,
(1998), wherein, the dioxygenases cleaves the aromatic ring via ortho-cleavage of .
ateohol
19
0 2-11ydroxy-Cis,cis-
muconate semialdehyde
Auto-oxidation 0.5%/day
NHOH
Imineralization
OH
OH
condensation products
catechol
Fig. 2.9: Interactions of aniline in the environment
0.4% per day
evaporation
Binding 4% percent
0
NHCCH3
2-hydroxy-muconate COOH
formate semialdehydehydrolase COOH
cis,eis-Muconate
CH2 COOH
cis-2-1-Iydroxypenta -2,4-dienoate
1 2-oxopent-4-enoate hydratase Maleylacetate
CO2
CH3 COOH
4-Hydroxy-2-oxovalerate
COOH i
Suecinic acid CH3
levulinic acid
4-hydroxy- 2-oxo-valerate aldolase
Pyruvate -I- Acetaldehyde
Chapter 11 Literature Survey
with amino group still present on the ring, to give 2-aminomuconic semialdehyde,
Enzyme deaminase replaces the amino group with hydroxyl group to give 2 -
oxocrotonic acid. Zhao et al., (2000), reported the transformation of 2-aminophenol
and 4-aminophenol by P. putida 2NP8 to respective intermediates 2- and 4-
iminoquinone which were further converted to quinones (Fig. 2.10). Further
downstream pathway followed the catechol or hydroquinone ring cleavage.
Degradation of 2 - and 4 - aminobenzoic acids follow two different pathways;
2-aminobenzoic acid could be transformed to 2,3-dihydroxy benzoic acid brought
about by anthranilate 3-monooxygenase seen in Pseudomonas sp. (Tanuichi et al.,
1964) or could be converted to catechol by anthranilate 1,2-dioxygenase in a fungus
Aspergillus niger (Kamath et al., 1990). P-aminobenzoate, similarly can be converted
to p-aminophenol by 4 -aminobenzoate hydroxylase in fungus Agaricus bn.sporu.s
(Tsuji et al., 1986), or converted to 3,4 dihydroxy benzoate by 4-aminobenzoate 3,4-
dioxygenase (Fig. 2.11). The catechol and hydroquinone formed are common
intermediates and is used by a variety of micro-organisms.
. p-Chloroaniline is degraded by Moraxella sp. following the pathway shown in
figure 2.12, as reported by Zeyer el al., (1985) via ortho -cleavage. Meta -cleavage
pathway of m-chloroaniline is reported to have followed by Comamonas testosterone
(Boon et aL, 2000) via 2,3 -dioxygenase pathway. Organism P. putida GJ31, able to
degrade 3-chlorocatechol was found to follow the proximal 2,3-dioxygenase (Mars et
al., 1997; Kaschabek et al., 1998) or distal 1,6-dioxygenase (Kaschabek et al, 1998)
(Fig. 2.13).
20
o-aminophenol
Aminophenol dioxygenase
NH2
NH 2-aminomuconic semialdehy de NH
COOH
CHO
Dehyfrogenase
NH2 0
1 —4 -i min quinone 2-imino tiinone
2-aminomueonic acid
COOH
COOH
Deaminase
2-oxalocrotonic COOH
acid
COOH
\If Deearboxylase
2-oxo-pent " COOH 4-enoate
Hy dratase
0 4-hydroxy -2-valerate COOH
OH
Catechol
OH 4-by droquinone
Fig. 2.10: Degradation pathway of aminophenols
HO
Aldolase and dehydrogenase Ring Cleavage Ring Cleavage
by hydroquinon path ay Pyruvate + acetyl CoA ,
2-aminobnezoate
anthranilate-3-monoxygenase anthranilate-1,2
-dioxygenase
2,3-dthydnaxy benzoate o-hydroxy benzoate
/ 4-aminobenzoate hydroxylase
COOH
OH p-hydroxy benzoate.
4-aminobenzoate 3,4-dioxygenase
COOH
OH OH
3,4-dihydroxy benzoate
NH2
4-aminobnezoate
Fig. 2.11: Degradation pathway of aminobenzoates
COOH
Fig.2.12: Degradation pathway of p-chloroaniline
NH2
COOH COOH
catechol CI ,2-dioxygenase
cis,cis-Muconate
aniline oxygenase
muconate cy clo-isomerase
O COOH COOH
l3-ketoadipate maleyl-acetate reductase
COOH COOH
dienelactone Maleylacetate hydrolase
COOH
° 0
Muconolactone
3 -oxoadipate CoA-tran sferase
O CSC0A COOH
Suecinyl CoA + Acetyl CoA acetyl-CoA C-acyltransferase
OH NH2 3-chloroaniline CI OH
1,2 dioxygenase C I
2-Chloro-hexa- 2,4-dienedioic acid
Cl-
Cl HOOCC OH OOH
HOOC coo
OH
hydroxymuconic acid chloride
CI COOH
ECHO
3-chloro,2-hydroxy mueonie senaialdehyde.
Cl -
hydratase
aldolase
Fig. 2.13: Degradation pathway of m-chloroaniline
3-Chloroaniline 3-Chloro-benzene-1,2-diol
distal extradiol cleavage I Proximal eatechol
extradiol 2,3-dioxygenase cleavage II
OH
TCA hydroxymuconic acid
itoxalocrotonate isomerase
Hoof O COOH "•••"/
4-oxalocrotonate
oxalocrotonate decarboxylase
2-oxopentanoate
OH 2-hydroxy oxovalcrate
TCA
pyruvatc + acetaldehyde
Chapter II Literature Survey
2.5. ENZYMES
2.5.1. Ovgenasea
Under aerobic condition, bacteria use oxygen not only as a terminal electron
acceptor but also to activate the aromatic compound for its breakdown and utilization.
Various enzymes produced by them catalyze the addition of oxygen to the aromatic
ring. These are widely called oxygenases. The difference between enzymes oxidases
and oxygenases is positional, non-specific addition of oxygen on the aromatic ring,
thus it would be difficult to predict the product formed, whereas the oxygenases add
oxygen atom at a fixed specific position in the ring.
Oxygenases can be classified based on the co-enzyme requirement or the
nature of the oxidizing substrate and the reaction products formed. Figure 2.14 shows
the classification of various oxidizing enzymes (http://us.expasy.org ).
2.5.1.1. Monooxygenaes
Monooxygenases catalyzes the reaction of addition of one oxygen atom in to
the aromatic ring. Based on the co-factors required for their recycling into active
form, there are four kinds. 1) NADH of NADPH dependent monooxygenases, 2)
Cytochrome P450 which involves an Fe(III) substrate, 3) Flavin-dependent
monooxygenases and 4) hydroxylases.
Reaction mechanism
The reactions carried out by these redox enzymes are simple. In case of
NADH of FADH dependent monooxygenases, the enzyme-substrate complex formed
uses molecular oxygen to oxidize the aromatic ring with a single oxygen atom while,
the other atom is reduced with the co-factor forming water.
21
Monooxygenases Dioxygenases
Phenol Oxidases Lipoxygenases
OXIDISING ENZYMES 02 serves as electron acceptor
Oxidases Uses 02 as electron acceptor
Produces H2O or H202
Peroxidases Uses H202 produce H2O
Oxygenases
Cytoch.rome P450 -
dependent
Flavin-dependent
Diverse Fe, Cu or Flavin-dependent
Other Monooxygenases
Chapter II Literature Survey
Substrate + donor-H + 02 H+ Substrate-0 + donor + H2O
Fig. 2.14: Classification of various oxidizing enzymes
Various reactions are catalyzed by the monoxygenases in transformation
processes; styrene monooxygenases oxidizes the ethene bond present on the side
chain of the benzene ring to form an epoxide called styrene oxide (Panke, 1999).
Wubbolt et al. (1994), has studied the function of xylene monoxygenases on the
conversion of toluene, xylene (Fig. 2.15) and certain non specific substrate such as
ethylbenzene to convert it to styrene oxide (Fig. 2.16a). Polyaromatic compounds
22
o-xylene
OH
Toluene oxygenase OH
benzyl alcohol
benzylalcohol dehydrogenase
benzaldehyde dehydrogenase
benzaldehyde
benzoic acid
catechol
COOH COON
OH
OH
COOH
OH
OH
toluene m-xylene p-xylene
Fig. 2.15: Action of monooxygenases on toluene and xylenes
• Ortho-pathway Meta-pathway Meta-pathway Ortho-pathway Meta-pathway
Benzo(a)pyrene-11 ,12-epoxide
Fig. 2.16: Styrene / ethylbenzene and benzo(a)pyrene oxidation by enzyme monooxygenase
a Styrene / ethylbenzene oxidation
HO
Styrene monooxygenase styrene oxide
isomerse photylacetaldehyde hydrogenise
styrene styrene oxide phenylacetaldehyde phenylacetate
/xylene oxygenase phenylacetate hydroxylase
Ethyl-benzene HO
b benzo(a)pyrene oxidation
Benzo(a)pyrene
0 0" 2-hydroxy phenylacetate
OH hydroxylase .1111•■■•••■■■■■..
gentisate
Benzo(a)pyrene-11,12-epoxide
0
OH
2-hydroxy phenylacetate
Chapter II Literature Survey
such as benzopyrene found in abundance in crude oil is affected by the enzyme
monooxygenase (benzo(a)pyrene 11,12 -epoxidase) and is converted to an epoxide
(Moody et al., 2004) (Fig. 2.16b). The epoxide formed is found to be toxic to humans
and the environment. Further hydroxylation of epoxide leads to the formation of
hydroxyl groups increasing its susceptibility towards ring cleavage. These are the
reactions brought about by NADH dependent monooxygenase. Monooxygenases can
also help in catalyzing various ringed ketone. One such example is conversion of
cyclohexanone to 1-oxa-2-oxocycloheptane (Mihovilovic et al., 2001) (Fig.2.17).
Buhler et al. (2002), has studied the catalyzation of the non-heme dependent
monooxygenase enzyme on the hydroxylation of a wide range of benzyl compounds
which includes toluene and xylenes and substituted toluenes and xylenes. These
compounds were converted to corresponding benzoyl alcohols. 2 -Hydroxyl biphenyl
was converted to 2,3 -dihydroxybiphenyls by enzyme 2 -Hydroxylbiphenyl 3 -
monoxygenase (Suske, 1997).
a) Cytochrome P450
Cytochrome P450 exists in most living creatures, including animals, plants,
and microorganisms, and plays an extremely important role in metabolism. These
enzymes are used to detoxify the chemicals by addition of a single oxygen atom in the
aromatic moiety. The mode of action of P450 is shown in fig 2.18 (Hata et al., 2005).
The monooxygenation reaction by P450 is initiated by the substrate binding to ferric
P450 (Fig.2.18(1)). When an electron (1 4 e) is introduced into substrate -bound P450,
the heme iron converts into the reduced form, Fe 2+ (2), and then an 02 molecule is
incorporated in the heme pocket (3). When another electron (2nd e) is introduced (4),
23
Fig. 2.17: Monooxygenation by eytoehrome P450 and enzyme monooxygenase
Cytochrome P450
5-hydroxy camphor
CH OH
H3C CH3 OH
terpineol
thioanisole
H3C CH3 OH
hydroxylated terpneol
0 Methanesulfinyl
benzene sulfoxide
Monooxygenase
cyclohexanone monooxygenase
cyclohexanone 1-oxa-2-oxocycloheptanc
Chapter II Literature Survey
the 02 molecule becomes very reactive and the substrate will be oxygenated by an
insertion of one 0 atom into the R—H bond (5). Hence, 0-0 bond cleavage should
occur after the introduction of the 2 nd e". The intermediates are, however, not able to
be observed in experiments because this reaction proceeds very quickly.
Fig. 2.18: The monooxygenase cycle by cytochrome P450.
(subastrate) R-011 Fe
(1)
(R-OH) Fe" 140
(5)
(R-H) Fel
at-H) Fe
(RH) Fe (0i
(4) at-in Fee 1011
Fruetel et al„ (1994) has studied some of the reactions carried out specifically by the
isolated enzyme cytochrome P450, they include the oxidation of camphor (terpene) to
hydroxylated camphor, a-terpineol to hydroxyl terpineol; thioanisoles to sulfoxides,
styrenes to epoxides (Fig.2.17).
24
Subunit. composition a a 0,3133
Prosthetic group FAD, [2Fe-2S]
[2Fe-2S] 3[2Fe-2S], 3FeII
OH OH 2H+
NAD(P)H + Oxidized Oxidized Oxidized
NAD(P)+ Reduced Reduced Reduced
2H+
Components Reductase NAp Feffedoxin NAp Oxygenase NAP
Chapter II Literature Survey
2.5.1.2. Dioxygenases
Dioxygenases is oxygen incorporating enzyme which is Fe-S dependent and
heme non-dependent. This enzyme catalyzes an addition reaction of two oxygen
atoms into the aromatic ring at vicinal positions. One of the well studied dioxygenase
enzymes is Naphthalene dioxygenase (Gibson and Parales, 2000). The mechanism of
the enzyme is shown in (Fig.2.19).
Fig 2.19: Mechanism of the naphthalene dioxygenase enzyme
Dioxygenases catalyzes the oxygen addition reaction to many aromatic
compounds followed by ring cleavage. One of the most common intermediates is
catechol, a simplest aromatic diols which is susceptible for ring cleavage. Ring
cleavage usually takes place around the two hydroxyl groups. The cleavage in
between the two hydroxyl groups (intradiol) is called ortho-cleavage (Cerdan et al.,
1994) while cleavage adjacent to the hydroxyl group is called (extradiol) meta-
cleavage (Ngai et al., 1990) (Fig.2.1), such a ring cleavage reactions is seen in
hydroquinone pathway (Eppink et al., 2000) (Fig.2.20), ben.zopyrene degradation
pathway
25
Fig. 2.20: Degradation pathway of 4-hydroxybe.nzoate/m-Hydroxy phenol
HO COON
OH
4-hydroxybenzoate
Hydroxylase (decarboxylating)
HO OH
hydroquinone
Hydroquinone hydroxylase
Hydroquinone hydroxylase
HO OH
3-hychexyphenol HO
1,2,4-trihydroxybenzene
Hydroxyhydroquinone dioxygenase
HOOC
HOOC
4-Hydroxy-hex2 -enedioic acid
Maleylacetate reductase
HOOC
HOOC
3-Oxo-hexanedioic acid
OH
Chapter II Literature Survey
(Moody et al., 2004) (Fig. 2.21), 3,4-hydroxyphenylacetate 2,3-dioxygenase (Arias-
Barrau et al., 2004) (Fig. 2.22). Dioxygenase also catalyzes reaction where in the
oxygen atoms are added on the side chain as in the case of conversion of tryptophan
to N-formyl kyurenine (Colabroy and Begley, 2005) (Fig. 2.23) cleavage reaction
catalyzed by 3-hydroxy anthranilate 3,4-dioxygenase (Fig. 2.8). Dioxygenase enzyme
action of addition of the oxygen atoms on naphthalene to give 1,2-dihydroxy
naphthalene (Barnsley, 1976) (Fig. 2.24) are some of the reactions catalyzed by this
enzyme.
2.5.1.3. Hydroxylases
Enzyme hydroxylases catalyze a reaction wherein it replaces a hydrogen atom
of the aromatic ring with a hydroxyl group. These enzymes are found to be
membrane-bound multiprotein complexes (Holland and Weber, 2000). Since the
activity of the protein is highest in its bound state, the mechanism of the enzyme has
not yet known as it could not be isolated.
Some of the reactions catalyzed by this enzyme include hydroxylation of 4-
hydroxybenzoate wherein decarboxylation followed by hydroxylation of the benzene
ring forms hydroquinone, fin-ther hydroxylation of hydroquinone or 1,3-
dihydroxybenzene forms 1,2,4-trihydrobenzene (Eppink et al, 2000) (Fig.2.20). Arias-
Barrau et al.; (2004), has shown the hydroxylation of phenylalanine to give tyrosine
(Fig. 2.22). Phenylalanine and tyrosine are both amino acids. Suzuki et al., (1991),
has shown the conversion of Salicyclaldehyde to catechol by salicylate hydroxylase
(Fig. 2.24).
The reactions carried out by cytochrome P450, monooxygenases,
hydroxylases explains the addition of a single oxygen atom in to the ring which could
26
benzo(a)pyrenc 4,5-dioxygenase
Benzo(a)pyrene
Benzo(a)pyrene-cis -4,5-dihydrodiol
OH
Hydroxy methoxy benzo(a)pyrene
4,5-chrysene- dicarboxyhc acid decarboxylasc
1 1 1,12-dihydroxy-benzo(a)pyrene methylIransfemsc
Fig. 2.21: Degradation pathway of benzo(a)pyrene
Benzo(a)pyrenc-11,12-epoxide
Benzo(a)pyrene-11,12-epoxide
epoxide hydrolase
OH
benzo(a)pyrene-trans-1 1,12- dihydrodiol dehydrogenase
OH
1 benzo(a)pyrene-cis4,5- dihydrodiol dehydrogenase
Dimethoxy benzo(a)pyrenc
OH H2N
phenylalanine
fnmerate + acetoacetate
4-hydroxy- phenyl -pyruvateoxidase
OH
HO
4-hydroxyphenylacetate
4-hydzoxyphenylacetate 3-hydroxylase
HO OH
1 4-hydroxy- phenyl-pyruvatedioxygenase
homogentisate pathway
HO
4-hydroxyphenylacetate 1-hydroxylase
Fig. 2.22: Degradation pathway of Phenylalanine/Tyrosine
1 4-hydroxyphenylacetate 3-hydroxylasc-2,3-dioxygenase
OH
OH
trans,cis-5-Carboxymethy1- 2-hydroxymuconate semialdehyde
Succinate + pyruvate
COOH
NH2
0 trYPtoPhan
NH 2,3 dioxygenase
N-formyl kynurenine
kynurenine forraamidase
OH
Fig. 2.23: Degradation pathway of Tryptophan
IrYPtoPhan
OH
NH2 0 NH2
kynurenine
kynurenine monoxygenase
HO OH
NH2 0 NH2
3-hydroxy kynurenine
kynureninase
3-hydroxy anthranilate 3,4 dioxygenase
Pyruvate + Acetate
OH
CHO Salicylaldehyde
Salicylate hydroxylase
Salleylate
Catechol
Fig. 2.24: Degradation pathway of Nathphalene
Naphthalene oxygenase Naphthalene
1,2- Dihydroxy Naphthalene
1,2-Thlydroxynaphthalene oxygenase
Salicylaldehyde dehydrogenase •
Chapter II Literature Survey
take place anywhere in the pathway both in biodegradative as well as during
synthesis. The reactions referred in this section explains the hydroxylation during
various degradative pathways. Hydroxylation of 2-hydroxyphenylacetate during
styrene degradation (Panke et al., 1999) (Fig. 2.16), kynurenine to 3-hydroxy
kynurenine (Colabroy and Begley, 2005) (Fig. 2.23), conversion of 4-
hydroxyphenylacetate to 3,4-hydroxyphenylacetate by 4-hydroxyphenylacetate 3-
hydroxylase (Arias-Barrau et al., 2004) (Fig. 2.22) are examples of rnonooxygenation
of aromatic compounds.
The incorporation of the oxygen atom in the aromatic ring plays an important
role in the degradation as it is this hydroxylation that makes it susceptible to microbial
attack. The introduction of two oxygen atoms makes it ready for the ring cleavage
(explained further) to give an aliphatic compound especially an acid or aldehyde
which is easily utilized by various organisms.
2.5.1.5. Laccases
These oxidases are included in the copper containing enzyme category. These
are involved in catalytic oxidation of diphenols and the major difference between the
two enzymes is that laccase has the ability to oxidize both o- and p-diphenols, where
as the enzyme phenol oxidase catalyzes reactions only with o-diphenols (Burton,
2003).
Laccases are blue multi-copper oxidases with four copper ions that are
coordinated with 3 redox sites. These have a molecular weight between 40000 to
140000 and are often produced as highly glycosylated derivates, where the
carbohydrate moieties increase their hydrophilicity and thus stabilize them in their
extracellular role.
27
Chapter II
Literature Survey
Reaction mechanism
Laccases initially reacts with the substrate forming a complex which then
reacts with molecular oxygen within the enzyme wherein, an intramolecular electron
transfer takes place leading to oxygen reduction. With the transfer of oxygen atom,
the product is released from the enzyme with liberation of water.
The substrates for the enzyme laccase includes methoxy phenol, phenols, n-
and p-diphenols, aminophenols, polyphenols, polyamines and lignin related
molecules. Demethylation reactions of lignin, ethoxy phenol acids and methoxy
aromatics, benzylamine conversion to benzaldehyde (Fig. 2.25), various
polymerization and co-polymerization of lignin molecules with phenols and
acrylamide are also catalyzed by laccases.
These enzymes in the presence of certain chemicals called mediators can
catalyze various reactions that are chemically not feasible. Mediators
(hydroxybenzotriazole (HBT); 2,2'-azinobis-(3-ethylbenzylthiozoline-6-sulphonate
(ABTS); 3-hydroxy-anthranilic acid (HAA)) are compounds that can undergo redox
reactions and in the process carry out reactions with high redox potential. Some of
these reactions involve the transformation of toluene and dimethoxytoluene to their
respective benzaldehydes.
Laccases also bring about coupling reactions using free radicals (Chignell,
1985) where the simple low molecular weight aromatic compound is converted to a
high molecular weight compound. Isouegenol is converted to dimeric products
(Baminger et al, 2001) and dimerization of substituted imidazole (Shuttleworth and
Bollag, 1986) (Fig.2.26a & b).
28
OCH3
CHO
COOH
OH
CHO
Veratryl alcohol
Fig. 2.25: Examples of typical laccase catalysed reactions
Ferulic acid Vanilic acid
Vanilin
CHO
Benzylimine Benzaldehy de
R
OCH3
Aromatic ketones
OCH3
Aromatic secondary alcohols
a
R - CH3 = Isougenol
R - CH2OH = coniferyl alcohol
OCH3
Radical formation
NHAc N
N---NHPr
...--0►
Substituted imidazole
NHAc 14
N--NHPr
+
b
NH2
Fig. 2.26: Polymerization reactions by laccases
..■
H3CO Dimers formed
dimeric imidazole product
Chapter II Literature Survey
2.5.1.6. Polyphenol oxidases
Polyphenol oxidases are also called enzyme tyrosinases as they hydroxylate
tyrosine with a hydroxyl group. They contain 2 copper ions at one reaction site in
each functional unit responsible for binding of both the molecular oxygen and the
substrate. The substrates are always phenol or dihyroxyphenol.
Phenol oxidases catalyses two reactions: 1) the hydroxylation of phenol to
catechol the enzyme is also called cresolase and 2) oxidation of catechols to quinones
the enzyme is called catecholase. There could be an extension of the reaction wherein
the quinones could polymerize to form melanin or give a polyphenolic compound.
The reaction mechanism proceeds in the following pathway; 1) the phenol
(substrate) is converted to a phenoxy radical, 2) Addition of the oxygen atom from
molecular oxygen. This oxygen is destabilized by polarization in the enzymes di-
copper active site, 3) oxidation reaction to convert catechol to quinone.
Polyphenol oxidases are involved in various substitution reactions of
replacement of hydrogen atom with the hydroxy group at the ortho position which is
not feasible through chemical synthesis (Fig. 2.27). Like laccases, these enzymes are
also involved in polymerization of aromatic compounds; the biphenol products
formed by the phenol oxidases reaction can be used in production of complex
compounds with from substrates such as flavonols having industrial importance
(Burton, 2003).
2.5.1.5. Peroxidases
Peroxidases are commonly found in plant, animal and microbial cells. These
are haeme protein containing Fe(LU) which undergoes redox reactions during the
29
Tyrosine
HO COOH
HO
L-DOPA
OCH3 OCH3
OH
Fig. 2.27: Examples of typical PPO catalysed reactions
NR2 NR2
OH
2-aminotetralins
NR2 NR2
OH
Chromamaines
p-hydroxyanisole
. A + H AH
A + OH
e AH
Chapter II Literature Survey
catalytic reactions (van de Velde et aL, 2001). These proteins react non-selectively via
free radical mechanism in the presence of hydrogen peroxide.
Colonna et al., (1999), proposed the mechanism of its catalytic cycle has been
shown if fig. 2.28. Compound-I is the oxidized state of enzyme peroxidase with ferry!
oxygen (radical) with two electrons. The ferryl state gets back to the ferric state by
two ways; 1) it gains two electrons, one each from two substrates or 2) oxidizes
hydrogen peroxide thereby gaining two electrons, thus showing catalase activity.
Fig.2.28: Catalytic mechanism of peroxidases.
Another form of peroxidase is isolated from the marine fungus Caldariomyces
fumago which catalyses the halogenation of the substrate. This enzyme was found to
be versatile as this could bring about catalysis of aromatic hydroxylation, epoxidation,
30
H2O
0 II
-CH3 21420 + 02 p-tol-S-CH3
Peroxidases
+ H202
Literature Survey Chapter II
sulfoxidation and catalase (Colonna, et al., 1999; van de Velde, et al., 2001). Figure
2.29 shows participation of peroxidase in various reactions.
Fig.2.29: substrates and products of enzyme peroxidases.
Ellipticinc quinonc iminc
2.6. ENVIRONMENTAL FACTORS
Various factors are responsible when biodegradation of the aromatic
compounds is considered: Some of the important factors include - positive
chemotactic behaviour of the bacterium towards the pollutant; bacterial ability to
produce specific enzymes; the nature of the pollutant and environmental factors (such
as oxygen (electron acceptor availability), pH, temperature, pressure and salinity.
31
Chapter II Literature Survey
2.6.1. Oxygen
2.6.1.1 Aerobic aromatic ring cleavage mechanism in bacteria
Bacterial response towards chemical substrates depends on various factors
depending on its environment. Polluted waters such as aquifers and submerged
sediment are normally anaerobic in nature. Bacterial interaction with contaminants
will change depending on the availability of oxygen in the environment, accordingly,
bacteria will follow aerobic or anaerobic type of degradation. Other factor equally
significant is the terminal electron acceptor. Oxygen is the widely used electron
acceptor under aerobic condition while Fe(III) also plays an equally important role.
Various aerobic reactions are catalyzed by enzynies such as oxygenises
(monooxygenases, dioxygenases), laccases, phenol oxidases and peroxidases.
2.6.1.2. Anaerobic aromatic ring cleavage mechanism in bacteria
Anaerobic environment harbours a completely different community of
microorganisms. These are found in aquifers, submerged sediments, polluted waters
and in environments where the oxygen levels are depleted. In such conditions
respiration of the organisms takes place with the terminal electron acceptors being
nitrates, sulfates, Fe (III), carbondioxide or other acceptors (Chlorate, Mn, Cr, U, etc)
(Diaz, 2004).
32
Chapter II Literature Survey
It was assumed that aromatic compounds would not be utilized under
anaerobic conditions but it is surprising to find that these compounds are being
utilized by ring cleavage pathway but follows a different set of reaction in the
pathway. Harwood and Gibson (1997), reviewed the possible pathways followed for
the degradation of aromatic compounds by various anaerobic bacteria. Fig.2.30 shows
an overall pathway followed by microorganisms which involves significant steps; 1)
CoA thioester formation, 2) ring reduction 3) introduction of a carbonyl group, 4) ring
opening, 5) b-oxidation sequence leading to the conversion of the remainder of the
molecule to acetyl-CoA.
Studies carried out by various researchers have shown that the aromatic
compounds carrying different substituents are primarily carboxylated and
thioesterified by anaerobic bacteria and which become available to be utilized by
various anaerobic bacteria. Van Schiel and Young (2000), has shown the initial
transformation of phenol to p-hydroxybenzoate by carboxylation in Thauera
aromatics K 172 (denitrifying bacteria), (via phenylphosphate and phenolate, to 4-
hydroxybenzoate by enzyme phenolcarboxylase intracellularly) followed by thio-
esterification, and ring cleavage. Hydroquinone was found to be anaerobically
degraded via gentisate in a sulfate-reducing bacterium Desulfococcus sp. HY5. The
carboxyl group of gentisate was thio-esterified and enzymatically transformed via ring
cleavage (Gorny and Schink, 1994).
A recent review carried out by Widdel and Rabus (2000), has shown that there
exists another mechanism by which the aromatic compounds are degraded. Amongst
the denitrifiers, fumerate is used as the terminal electron acceptor. Aromatic
compounds with alkyl chain such as toluenes, cresols, xylenes, cymenes, methyl
naphthalenes
33
OH
3-Chlom benzoic acid
0
O conmarate
tocatechuate
vanillate
CoASOC
CoASOC
CoASOC
Fig. 2.30: Anaerobic degradation pathway of the benzene ring
CoASOC
4-hydroxybenzoyl-CoA
benzoyl CoA reductase
CoASOC
benzoyl-CoA
eyelohex-1,5-diene-1-earboxylCoA
CoASOC
cyclohex-2,5-diesie-1-carboxylCoA
CoASOC
HO 3-hydroxy Pimelyl-Co 2-keto- cyclohexane-l-earboxylCoA Pimelyl-CoA
2-hydroxy cyclohexane-l-carboxylCoA
HOOC
HOOC pinadyi_aaA CoASOC 2-keto- cyclohexane-l- carboxylCoA hydrolase CoASOC hydroxylase
2-hydroxy cyclohexane-l-earboxylCoA dehydrogenase
CoASOC
cyclohex-1,-ene-l-carboxylC,oA HO
CoASOC
6-hydroxy-cyclohex-1-ene-1-earboxylCoA
cyclohex-1,-ene-l-carboxylCoA hydratas.
HO
AcetylCoA
4-hydroxybenzoate
benzoate CoA ligase
=Or
Toluene
COOH
0'
HO
OH
OH
ma•
2-aminobenzoate
cyclohex-1,4-diene-l-carboxylC,oA
Chapter II Literature Survey
(Beller et al., 1992; Muller et al., 2001; Achong et al., 2001; Safinowski and
Meckenstock, 2004) as couples with fumerate to give benzylsuccinate derivatives
which are then converted to benzoate. On the other hand, aromatics such as ethyl
benzene, propyl benzene showed their transformation to alcohols and naphthalene to
2-naphthanoic acid. The naphthalene ring cleavage pathway brought about by
naphthalene degrading bacteria is proposed by Annweiler et al., (2002), is shown in
Fig.2.31.
2 6.2. Temperature
2.6.2.1. High temperatures
Natural environments with high temperatures are observed at hot water
springs, areas around active volcanoes, hydrothermal vents in deep oceans. Various
other environments include composts, industrial storage areas containing utilizable
effluents. The organisms growing optimally at high temperatures are called
thermophiles while thermotolerant organisms consists of those that grow optimally at
lower temperatures but can tolerate higher temperatures. Some of the thermophilic
bacteria growing at moderate temperatures belong to genus Bacillus, Geobacillus,
Thermoactinomyces, Clostridium, Thernzoanaerobacter, Thermoplasma; extreme
temperatures -Thermus, Therrnodesulfobacterium, Sulfolobus, Thermomicrobium,
Dictyoglomus, Methanccoccus, Sulfitrococcus, Thermotoga;, hyperthermophiles -
Meihanoccus, Acidianus, Archaeoglobus, Methanopyru, Pyrobaculum, Pyrococcus
and Thermococcus.
Various PAH compounds such as fluorene, fluorathene, phenanthrene are
considerably less soluble at mesophilic temperatures and Viamajala et al. (2007) have
shown that there is an increase in the solubility with an increase in temperature and
34
COOH 5,6,7,8-tetrahydro-2-napbthoic acid
Naphthalene
COOH
tetralin
oxidation C001
- OH [ hydroxydecahydro-
2-naphthoic acid
C00
0
1-1
Fig. 2.31: Anaerobic degradation pathway of naphthalene/methyl-naphthalene
hydrogenation
octahydro-2-naphthoic acid (position of the double bond is unknown)
COOH oci,COON
hydration ,1 decahydro-2 -naphthoic acid
SCoA ring cleavage + Q oxidation
COOH
13 oxidation
COOH
[ f3-oxo-decahydro-2 -naphlhoic acid
1111604-diaeid
COON
COOH
cis-2-carboxycyclo hexylacetic acid
Chapter II Literature Survey
has claimed to have isolated a thermophile Geobacilli from a compost which utilized
phenanthrene. Phenol transformation to catechol followed by meta-cleavage was
observed in. Bacillus stearothermophilus (Kim and Oriel, 1995). Biodegradation of
PCB by a thermophile, isolated and chracterised by Kimbara (2005), following the
similar pathway with the addition of a dihydroxy group at 2,3 positions, followed by
ring cleavage by dioxygenases.
2.6.2.2. Low temperatures
Psychrophilic bacteria include microorganisms that are able to grow optimally
at low temperatures in the Antic, Antarctica and cold areas where there is formation of
ice. Anthropogenic activities at the Arctic region is seen when petroleum products are
used for heating and other mechanical activities leads to oil spills on ice. Transport of
pollutants from other parts of the world which occur due to natural calamities and
other anthropogenic activities to the Arctic regions by water currents is possible.
Thus, it is of significance to understand the microbial population surviving in these
conditions and their biodegradation activities. Mohn and Stewart (2000), studied the
limiting factors for the biodegradation of hydrocarbons in these conditions. It has
been found that the soil in the Arctic is barely seen when the ice melts during
summer, the permafrost prevents movement of water which creates an active zone
where most of the hydrocarbon exists and probably the site showing highest activity.
Mohn also reported that the psychrotolerant bacteria, instead of psychrophilic, are the
dominating population showing highest activity at higher temperatures Le. at 15 —
20°C. This may suggest that the hydrocarbons become available to microbial attack at
a higher temperature. Additional nutrients added such as nitrogen or heavy metals
retard their ability to degrade the hydrocarbons. It is thus evident that bacteria bring
35
Chapter II Literature Survey
about degradation of aromatic compounds at such cold temperatures. Eriksson et al.
(2001), showed a similar reaction where temperature plays an important role in
hydrocarbon degradation. Studying the degradation at temperatures of 7, 0 and -5°C,
hydrocarbon degradation was evident at a higher temperature. Eriksson et al. (2003),
enriched a microbial population belonging to genus Acidovorax, Bordetella,
Pseudomonas, Sphingomonas, and Variovorax from Arctic soil able to degrade PAH
at 7°C anaerobically with nitrate as electron acceptor. The efficiency was 39%
removal of 2-methylnaphthalene and tluorene at 7°C and up to 80% removal at 20°C
under aerobic conditions after an incubation period of 90 days. PCBs have also been
found at the Arctic and their degradation have been studied by Master and Mohn
(1998), where isolates closely belonging to the genus Pseudomonas could breakdown
PCB at 7°C, and their removal at higher temperatures were much higher (90% at
37°C) .
2.6.3. Salinity
One of the natural extremities includes the environments with high salinity
Salinity varies with ecosystems, fresh water ecosystem shows negligible or no salinity
whereas open seas show salinity of 30-35% salt concentration. Salt pans have salt
concentration at saturation levels. Estuarine ecosystem shows a fluctuation in salt
concentration due to constant mixing of sea water with fresh water. The general term
given to the organisms that survive, tolerate and thrive in such environment is
Halophiles which are included in a separate domain called Archaea (Woese et al.
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Chapter II Literature Survey
1990), whereas organisms that can tolerate high salt concentrations but can even grow
at environments with no or less salt concentrations are called halotolerant which
belong to eubacterial domain. Some of the members of halophiles that were isolated
from Dead Sea belonged to the family Halobacteriaceae, such as genus Haloferax,
Haloarcula, Halorubrum (formerly Halobacterium) (Arahal et al. 1996). Pagie et al.
(2005), have shown that bacteria belonging to some of the genera mentioned above
thrive in salterns. These organisms belonged to obligate halophiles as they could not
survive in medium at salt concentration below 15%. Halophilic archaea maintain an
osmotic balance with the hypersaline environment by accumulating high salt
concentrations, which requires salt adaptation of the intracellular enzymes (Ventosa et
al., 1998; Hough and Danson, 1999; Oren, 1999). Halophilic bacteria have adapted to
the osmotic stress of high-salinity environments by actively accumulating K+,
glycerol, betaine and ectoine within the cytoplasm (Peytona et al., 2002; Woolard and
Irvine, 1995; Galinski et aL, 1985; Jebbar et al., 1992). Eubacteria are more
promising degraders than archaea as they have a much greater metabolic diversity.
Their intracellular salt concentration is low, and their enzymes involved in
biodegradation may be conventional (i.e. not salt-requiring) enzymes similar to those
of non-halophiles. The use of microorganisms able to degrade organic wastes in the
presence of salt could prevent costly dilution to lower the salinity, or the removal of
salt by reverse osmosis, ion exchange or electrodialysis before biological treatment.
Thus these organisms play a vital role in the biodegradation and biomineralization of
organic residues in saline environment. Nicholson and Fathepure (2004), has reported
to have isolated a mixed population of halophiles from a brine soil which was
dominated with the culture Marinobacter spp. The enriched cultures were tested to
check their ability to degrade benzene, toluene, ethylbenzene or xylenes (BTEX
37
Chapter II
Literature Survey
components). This culture had an ability to degrade all the components when
introduced as pure compounds in the growth medium at a concentration of 20-301.tmol
at salt concentration of 2.5 M and at 30°C. It was observed that the enriched culture
could degrade toluene the fastest (within a week) while benzene, ethylbenzene, or
xylenes required roughly 2 to 3 weeks. Emerson et al. (1994), have isolated a
halophile Haloferax D1227 that utilized aromatic compounds such as benzoate,
cinnamate, and phenylpropanoate in the presence of 1.7-2.6 M NaCI at 45°C. Garcia,
et al., (2004), have reported to have isolated a halophile Halomonas organivorans a
moderate halophile able to utilize a variety of aromatic compounds which includes
benzoic acid, p-hydroxybenzoic acid, cinnamic acid, salicylic acid, phenylacetic acid,
phenylpropionic acid, phenol, p-coumaric acid, ferulic acid and p-aminosalicylic acid.
2.6.4. pH
2.6.4.1. Acidic pH
Naturally occurring acidic environments are present in various mining areas.
These natural acidic conditions seem to have formed due to a combination of bacterial
and aerobic oxidation process. The acidic condition arise in areas rich in pyrite ore.
These are a kind of sulphide ore of iron, copper or zinc or a mixture of all three
depending on the abundance of the ore. Chemical oxidation is instantaneous wherein
the pyrites react readily with oxygen to give metal ions with a release of acid, mostly
H2 SO4. Further, the reactions are catalyzed by the acidophilic bacteria present most
common being Thiobacillus jferroxidans, which oxidize the metal ions to serve as a
source of energy within the cell. The oxidized metal inturn release the sulphur from
the ore giving rise to more metal ions and more acid and thus the reactions become
self sustaining and ongoing. In these conditions, the acidic products containing metals
38
Chapter II Literature Survey
of iron of respective ore is released to an extent that it pollutes the entire aquifer that
comes in contact or could percolate down into the ground water thereby polluting it.
Various iron ore mines, coal mines etc. can have such acidic conditions provided they
have ore pyrites present. Mines not having such ore pyrite may not develop an acidic
environment. The iron oxidizing strict acidophilic bacteria can also be detected in acid
springs. Sulfolobus is another iron oxidizing bacteria detected in hot acid springs.
Gonzales-Toril et al., (2003), have reported that Tinto river in southwestern Spain is
highly acidic with pH of 1.5-3.1 and has a high concentration of metal ions; iron
contributes to a concentration up to 20g/lt, copper up to 0.7g/it and zinc up to 0.56g/lt.
which was due to naturally occurring metal pyrites upstream. Phylogenetic 16s rRNA
analysis of the water body showed the presence of organisms related to Leptospirillum
spp., Acidithiobacillus .ferrooxidans, Acidiphilium spp., "Ferrimicrobium
acidtphiltun," Ferroplasma acidiphilum, and Thermoplasma acidophilum, microbes
belonging to iron oxidizing group. More than 80% of the cells were affiliated with the
domain Eubacteria, with only a minor fraction corresponding to Archaea. Other
thermophilic bacteria belonging to genus Acidisphaera spp., Acidiphilium and
Acidithiobacillus spp. were isolated from acidic soil samples (pH ranging between
2.8-3.8) rich in sulfates in Yellostone national park, Montana, USA, which had a
natural seep of hydrocarbons containing hexadecane (Namamura et at, 2005).
Stapleton et al. (1998), have reported the presence of indigenous acidophilic bacteria
surviving at pH 2.0 at a runoff of a longterm storage in a coal-pile basin, that brings
about 40% oxidation of aromatic compounds like naphthalene and toluene to carbon
dioxide, thereby suggesting that the biodegradation can occur at acidic pH.
39
Chapter 11 . Literature Survey
2.6.4.2. Alkaline pH
The pH plays an important role in the degradation of any compound. Optimum
level of degradation is observed in neutral condition where the neutrophilic bacteria
are abundant and gets acclimatized to the new introduced pollutant. pH is an
important factor as it determines the ability of compound to dissociate. It has been
observed that certain organic compounds such as chlorophenols and nitrophenols
dissociate and their toxicity lowered with increase in pH (Holcombe et al., 1980;
Kishino and Kobayashi, 1995; Kulkarni and Chaudhari, 2006) and makes them
vulnerable to microbial attack leading to its degradation. The most important criteria
for degradation of such organic compounds is ability of microbes to survive and grow
in such alkaline environments. The most stable naturally occurring alkaline
environments are soda lakes with pH values up to 11.5. They are located in areas
characterized by a unique combination of geological, geographical, and climatic
conditions that diminish the significant buffering capacity of atmospheric CO2 by the
evaporative concentration of sodium carbonate (Kleinsteuber et aL, 2001). The soda
lakes in the Rift Valley of Kenya and similar lakes found in other places on Earth are
highly alkaline with pH values of 11 to 12. The Kenyan-Tanzanian Rift Valley
contains a number of soda lakes whose development is a consequence of geological
and topographical factors. The salinities of these lakes range from around 5% total
salts (w/v) in the case of the more northerly lakes (Bogaria, Nakuru, Elmentiata and
Sonachi) to saturation in the south (Magadi and Natron) with roughly equal
proportions of Na2CO 3 ,and NaCI as the major salts in some of the natural alkaline
environments (Ulukanli and Diurak, 2002). Isolation of bacteria from environments
otherwise hostile to neutrophilic bacteria has been reported. These organisms
requiring alkaline condition as a prerequisite for their growth are called
40
Chapter II Literature Survey
alkalophiles/alkaliphiles and are able to tolerate pH values in a range of 8.0 to 12.0.
Based on their optimal growth requirements, they are called as obligate alkalophiles
(pH values 10.5-12.0) and alklotolerant (tolerates pH from 8.0 to 12.0 but has an
optimal pH requirement of 8.0). Alkalophiles isolated from Magadi in Kenya
belonged to the family Halomonadaceae grouped under i-proteobacteria. These
isolated bacteria were grouped under genus Halomonas, the various species that were
isolated were H. elongata, H. halodenitrifican.s, H. desiderata, H. cupids and H.
magadii (Duckworth et al., 2000). Horikoshi (1991), had reported that the majority of
bacteria belong to genus Bacillus, Micrococcus, Corynebacterium, Pseudomonas,
Flavobacterium, Actinomycetes such as Streptomyces, Nocardiopsis and Yeasts.
These belonged to the bacteria group that did not grow in the presence of high
concentration of salt. Whereas, the species belonging to Halomonas grew well in the
presence of salt. These were also called halotolerant alkalophiles. Alkalophiles were
isolated from other parts of the world such as Halomonas organivorans were isolated
from Spain (Garcia et al., 2004), Halomonas boliviensis from Bolivia (Quillaguaman
et al., 2004), H. koreensis from Korea (Lim et aL, 2004). Halomonas species were
also found active in cold conditions at Antartica and was named as Halomonas glade
by Reddy et al., (2003). Another group of microorganisms growing in such an
environment with high alkalinity and high salt concentration as a prerequisite was
isolated and called Haloalkaliphiles. Well known archaea Natronococcus and
Natronobacterium, were isolated from the salt lake from Magadi, Kenya (Tindall et
al., 1984; Mwatha and Grant, 1993). Bacteria belonging to genus Halomonas seem to
share the salt tolerating characteristics with halophiles. Halotolerant alkaliphilic
bacteria as mentioned by Garcia (2004), have the capability to degrade many aromatic
41
Chapter II Literature Survey
hydrocarbons. The versatile nature of these bacteria makes it unique and can be
utilized to degrade various pollutants under extreme conditions.
2.7. ROLE OF BIOSURFACTANTS IN BIODEGRADATION OF AROMATIC
COMPOUNDS
A large number of these compounds are insoluble and therefore not easily
available for degradation/transformation by the microorganisms. The non-availability
of the aromatic compounds could also be attributed to their adsorbed state to the soil
particulate matter or other surfaces. Some of the hydrophobic compounds include
aromatic and aliphatic hydrocarbons, resins, tars, etc., which naturally source from
crude oil. These also include xenobiotic compounds released in nature through
anthropogenic activities.
Bacteria have developed a strategy of utilizing such compounds to facilitate
biodegradation by different mechanisms. Mechanisms so far known are production of
biosurfactants, bioemulsifiers and direct cell-substrate attachment by the organisms. A
few examples are elaborated here.
Microorganisms produce a variety of surface-active agents (or surfactants)
which lower surface and interfacial tensions efficiently to allow emulsification with
less energy and bind tightly to surfaces thereby stabilizing the emulsions. This
facilitates the availability of the non-available compounds for degradation. The term
'biosurfactant' has been often used loosely to refer to other compounds like
biopolymers which generally do not reduce interfacial tension but may prevent oil
droplets from coalescing (Hommel, 1990). Biosurfactants also play an important role
in regulating the attachment-detachment of microorganism to and from surfaces,
which can increase aqueous dispersion of insoluble compounds by many orders of
42
Chapter II Literature Survey
magnitude, thereby increasing their chances of being degraded. In addition,
emulsifiers are also involved in pathogenesis, quorum sensing and biofilm (Ron and
Rosenberg, 2001).
Biodegradation requires uptake of the substrate by the cells, which in turn
requires contact between the substrate and the cell. Contact is determined by two
factors: (1) available substrate surface area and (ii) affinity of microbial cells for the
substrate. Biosurfactants increase dispersion or surface area for microbial attachment,
which increases biodegradation (Zhang and Miller, 1994). These can be divided into
low-molecular-weight molecules that lower surface and interfacial tensions efficiently
(Cooper and Zajic, 1980) and high-molecular-weight polymers that bind tightly to
surfaces (Rosenberg and Ron, 1997). These are generally glycolipids which includes
rhamnolipids (Al-Tahhan et al, 2000), trehalolipids (Li et al., 1984) and sophorolipids
(Cooper and Paddock, 1983). Some of the common high-molecular-weight
compounds include emulsans, alasan, liposan mainly composed of polysaccharides,
proteins, lipopolysaccharides, lipoproteins or a complex mixture of these biopolymers
(Rosenberg and Ron, 1999).
Biosurfactants are a structurally diverse group of surface-active molecules
synthesized by microorganisms. While the bioemulsifiers allow easy mixing between
the two immiscible phases, biosurfactants stabilizes the emulsion formed. They have a
unique amphipathic property derived from their complex structures, which include a
43
Chapter II Literature Survey
hydrophobic/lipophilic portion (usually hydrocarbon (alkyl) tail of one or more fatty
acids which may be saturated, unsaturated, hydroxylated or branched) attached to
hydrophilic group by a glycosidic, ester or amide bond. Biosurfactants form spherical
or lamellar micelles when surfactant concentration exceeds a compound-specific,
critical micelle concentration (CMC). Hydrophobic compounds becomes solubilized
in the hydrophobic cores of the micelles, which leads to the transfer of hydrophobic
compounds from solid, liquid or sorbed state in to the water phase. There are
furthermore reports of species-specific and energy-dependent uptake of biosurfactant-
solubilized compounds, which points at a direct interaction of biosurfactant micelles
with cell membranes (Beal and Betts, 2000 and Noordman and Janssen, 2002). Seeing
that many biosurfactants represent constituents of cell envelopes (Neu, 1996), the
possibility of a fusion between micelles and cells is indeed not far-fetched.
Utilization of biosurfactants has also been reported for mechanisms such as
detachment form a substrate seen during conditions 1) depletion of utilizable carbon
source from a mixture of compounds so that the cells could attach to another droplet
of liquid containing the substrate or 2) depletion of oxygen level at the bottom of the
biofilm (Neu, 1996).
A recent finding indicates the existence of horizontal transfer of high-
molecular-weight emulsifiers from the producing bacteria to heterologous bacteria.
Alasan, the exocellular polymeric emulsifier produced by A. radioresistens KA53,
was shown to bind to the surface of Sphingomonas paucimobilis EPA505 and A.
calcoaceticus RAG-I and change their surface properties. The transfer could be shown
after incubation of the recipient cells with the purified emulsifier (Osterreicher-Ravid
et al., 2000). This horizontal transfer of bioemulsifiers from one bacterial species to
44
Chapter II Literature Survey
another has significant implications in natural microbial communities, co-aggregation
and biofilms formation.
2.8. ROLE OF EPS IN BIODEGRADATION OF AROMATIC COMPOUNDS
Besides surfactants, bacteria producing film called biofilm overcome the
problem of utilization of insoluble compounds by producing exopolymeric substances
(US) also known as exopolysaccharides. Such an approach has been utilized to leach
in the insoluble or immiscible compounds present in the environment as free or as
surface sorbed compounds. Various authors have studied the role of EPS in utilizing
insoluble aromatic compound in nature by bacteria (Moreno et al., 1999; Obuekwe
and Al-Muttawa, 2001; Janecka et al., 2002; Rodrigues et at, 2004).
Exopolysaccharides are high molecular weight organic macromolecules
formed by polymerization of similar or identical building blocks, which may be
arranged as repeating units within the polymer molecule. EPS may contain non-
polymeric substituents of low molecular weight, which greatly alter the structure and
physiochemical properties. The extracellular polysaccharide could compose of
various sugars, amino sugars, sugar acids and carry organic substituents such as
acetyl, succinyl or pyruvyl groups and other inorganic substituents such as phosphates
and sulfates (Table 2.1). Proteins interact with polysaccharide compounds to form
glycoproteins by glycosylic bonds or can be substituted with fatty acids to form
lipoproteins (Wingender et al., 1999).
These characteristics produce a wide heterogeneity among the polymer, thus
representing a rich source of structurally diverse molecules with unique physical and
chemical properties. Many of the polysaccharides are relatively soluble, and because
of their large molecular mass, yield highly viscous aqueous solutions. As the presence
45
Chapter II Literature Survey
of uronic acid moieties confer a net negative charge, pyruvate residues contribute to
the water binding property (Decho, 1990). However, polysaccharides such as
hyaluronic acid can bind up to 1 kg water (gm polysaccharide) -1 . It is probable that
many of the EPS in biofilms bind lesser quantities whilst some, like bacterial
cellulose, mutan or curdlan, manage to exclude most water from their tertiary
structure. Hydrophilicity is also dependent on their composition and their tertiary
structure (Neu and Poralla, 1988). Authors have also reported in 1990 that the sugar
monomers of polysaccharides can be hydrophobic or hydrophilic depending on the
degree of hydroxylation: And some of the polymers contain both hydrophilic as well
as hydrophobic regions.
Interaction of ions has been observed with the carboxylic groups on the EPS
to yield networks of macromolecules which showed increased viscosity or gelation.
Various cations may compete for the same binding site, as was shown by Loaec et al.
(1997), Adsorption of heavy metal ions to the polymer has been observed by various
authors (Farres et al., 1997; Ferris et al , 1989; Geddie and Sutherland, 1993;
Mittleman and Geesey, 1985) .
The EPS also contributes to the mechanical stability of the biofilms (Mayer et
al., 1999), enabling them to withstand considerable shear forces. Other structural
properties of the polymer like rigidity or flexibility is based on the type of bonding in
between the chains of the polymer; 1,4-0- or 1,3- 13- linkages confers considerable
rigidity, as seen in the cellulosic backbone of xanthan from Xanthomonas campestris.
1,2-a- or 1,6—a— yields ,more flexible structures. The presence of 0-succinyl esters,
0-acetyl or pyruvate ketals in the polysaccharide gives a varied conformation —
46
Chapter II Literature Survey
forming random coils or helicals. The long polysaccharide chain involves in a number
of interactions between themselves and the substrate.
Due to the presence of the hydrophobic regions in the polymer made up of
high glucosyl residue, it has an ability to desorb the insoluble compounds from the
dormant surface in to itself thus making it available for the bacteria for mineralization
and utilization.
Literature survey has shown that various studies have been carried out on
biodegradation of aromatic compounds or substituted aromatic compounds containing
nitro and amino groups. All these studies reported are under normal neutral conditions
as such. Only a few reports are available on degradation of aromatic amines under
alkaline condition. The present study was therefore been undertaken with a view to
understand the interactions of aromatic amines under alkaline conditions with bacteria
able to grow at pH 10.5.
47